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Identification of S-Nitrosylation Motifs by Site-Specific Mapping of the S-Nitrosocysteine Proteome in Human Vascular Smooth Muscle Cells

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Identification of S-Nitrosylation Motifs by Site-Specific Mapping of the S-Nitrosocysteine Proteome in Human Vascular Smooth Muscle Cells Identification of S-nitrosylation motifs by site-specific mapping of the S-nitrosocysteine proteome in human vascular smooth muscle cells Todd M. Greco*, Roberto Hodara*, Ioannis Parastatidis*, Harry F. G. Heijnen†, Michelle K. Dennehy‡, Daniel C. Liebler‡, and Harry Ischiropoulos*§ *Stokes Research Institute and Departments of Pediatrics and Pharmacology, Children’s Hospital of Philadelphia and University of Pennsylvania, Philadelphia, PA 19104; †Thrombosis and Haemostasis Laboratory, Department of Cell Biology, University Medical Center Utrecht, and Institute for Biomembranes, 3584 CH, Utrecht, The Netherlands; and ‡Department of Biochemistry and Mass Spectrometry Research Center, Vanderbilt University School of Medicine, Nashville, TN 37232 Edited by Louis J. Ignarro, University of California School of Medicine, Los Angeles, CA, and approved March 22, 2006 (received for review January 27, 2006) S-nitrosylation, the selective modification of cysteine residues in critical roles for nitric oxide, the targets of S-nitrosylation in proteins to form S-nitrosocysteine, is a major emerging mechanism vascular smooth muscle cells are largely unknown. To that end, by which nitric oxide acts as a signaling molecule. Even though proteomic approaches are highly informative in providing a global nitric oxide is intimately involved in the regulation of vascular assessment of the modified proteins in cells and tissues. smooth muscle cell functions, the potential protein targets for Proteomic approaches based on the biotin-switch method have nitric oxide modification as well as structural features that underlie been used to identify potential targets of S-nitrosylation in various the specificity of protein S-nitrosocysteine formation in these cells model systems including murine brain tissue (15) and RAW 264.7 remain unknown. Therefore, we used a proteomic approach using cells (16), Mycobacterium tuberculosis (17), mouse mesangial cells selective peptide capturing and site-specific adduct mapping to (18), and human aortic endothelial cells (19, 20), yet the structural identify the targets of S-nitrosylation in human aortic smooth features that subserve the specificity of S-nitrosylation remain muscle cells upon exposure to S-nitrosocysteine and propylamine contentious. Recently, a peptide capture approach simultaneously propylamine NONOate. This strategy identified 20 unique S-ni- identified 68 unique S-nitrosocysteine residues belonging to 56 trosocysteine-containing peptides belonging to 18 proteins includ- proteins from S-nitrosoglutathione-treated rat cerebellar lysates ing cytoskeletal proteins, chaperones, proteins of the translational (21). Analysis of the identified peptides by a machine learning machinery, vesicular transport, and signaling. Sequence analysis of approach did not reveal linear sequence motifs under the experi- the S-nitrosocysteine-containing peptides revealed the presence of mental conditions used (21). However, subsequent inspection of the acid͞base motifs, as well as hydrophobic motifs surrounding the identified peptides indicated a prevalence for an acid͞base motif, identified cysteine residues. High-resolution immunogold electron suggesting that exploration of additional S-nitrosocysteine pro- microscopy supported the cellular localization of several of these teomes may further clarify the structural motifs that underlie the proteins. Interestingly, seven of the 18 proteins identified are specificity of S-nitrosylation. localized within the ER͞Golgi complex, suggesting a role for To this end, in intact human aortic smooth muscle cells S-nitrosylation in membrane trafficking and ER stress response in (HASMC) exposed to S-nitrosocysteine (CysNO) or propylamine vascular smooth muscle. propylamine NONOate (PAPANO), we identified potential targets of S-nitrosylation and evaluated S-nitrosylation motifs under con- nitric oxide ͉ proteomics ͉ S-nitrosothiols ditions that preserve the cellular localization of proteins as well as endogenous protein–protein interactions. Using a proteomic ap- proach that selectively identified the modified S-nitrosocysteine -nitrosylation, the formal transfer of nitrosonium to a reduced residues, 18 proteins were identified. The localization of several of cysteine, is a reversible and selective posttranslational modifi- S these proteins was further supported by high-resolution immuno- cation that regulates protein activity, localization, and stability, and gold electron microscopy. Primary sequence analysis of the S- also functions as a general sensor for cellular redox balance (1–7). nitrosocysteine-containing peptides revealed the presence of acid͞ The formation of protein S-nitrosocysteine requires the removal of base motifs as well as the occurrence of cysteine residues within a single electron, i.e., the conversion of the nitrogen in nitric oxide hydrophobic pockets. from an oxidation state of 2 to 3. Several distinct pathways could satisfy the formation of protein S-nitrosocysteine adducts in bio- Results and Discussion logical systems, such as autooxidation of nitric oxide forming higher Formation of S-Nitrosocysteine Protein Adducts in Human Aortic oxides of nitrogen, radical recombination of thiyl radical with nitric Smooth Muscle Cells. The intracellular protein S-nitrosocysteine oxide, catalysis by metal centers, the direct reaction of nitric oxide content was evaluated by reductive chemistries coupled with chemi- with a reduced cysteine followed by electron abstraction, and luminescence detection (22). Naı¨ve HASMC in culture had levels transnitrosation reactions carried out by S-nitrosoglutathione, of protein S-nitrosocysteine below the lower limits of detection, and other small molecular mass S-nitrosothiols, and more recently, Western blot analysis failed to document expression of nitric oxide S-nitrosocysteine-containing proteins (8–10). synthases in these cells (not shown). Therefore, to generate endog- In vascular smooth muscle cells, nitric oxide derived from enous S-nitrosylated proteins, intact cells were exposed to either endothelium regulates important biological functions beyond re- laxation, such as phenotypic changes, proliferation, and commit- ment to undergo apoptosis (11, 12). Previous studies have shown Conflict of interest statement: No conflicts declared. that the molecular mechanisms underlying the functions of nitric This paper was submitted directly (Track II) to the PNAS office. oxide in vascular smooth muscle are mediated by both soluble Abbreviations: HASMC, human aortic smooth muscle cell; CysNO, S-nitrosocysteine; PA- guanylate cyclase-dependent and independent mechanisms (12– PANO, propylamine propylamine NONOate; HPDP–biotin, N-[6(Biotinamido)hexyl]-3Ј-(2Ј- 14). It has been suggested that selective S-nitrosylation of protein pyridyldithio) propionamide; MS͞MS, tandem MS. targets are responsible for the guanylate cyclase-independent reg- §To whom correspondence should be addressed. E-mail: [email protected]. ulation of vascular smooth muscle cell biology (13). Despite these © 2006 by The National Academy of Sciences of the USA 7420–7425 ͉ PNAS ͉ May 9, 2006 ͉ vol. 103 ͉ no. 19 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0600729103 Downloaded by guest on October 1, 2021 Fig. 1. Evaluation of SEQUEST peptide assignments. (A) An MS͞MS spectrum (XCorr 3.6) assigned to an S- nitrosocysteine-containing peptide from 14-3-3 protein ␨ that met all selection criteria and was accepted. (B)An MS͞MS spectrum (Xcorr 4.1) assigned to a peptide from vimentin. Although this assignment passed the initial selection criteria, it was ultimately rejected because the top three most intense fragment peaks were not as- signed (arrow). The evaluation of SEQUEST peptide assign- ments was assessed by multiple selection criteria as fol- lows: (i) Only peptide assignments that identified a biotin-HPDP derivitized cysteine (ϩ428) included in the y-orb-ion series were considered. (ii) Each experimental condition was performed in quadruplicate, with peptide assignments evaluated if they appeared in at least three of the four independent replicates. (iii) Peptide assign- ments that passed these two selection filters were then evaluated by output scores assigned by SEQUEST and were rejected if they did not meet specific threshold values as described in the Materials and Methods.(iv) If peptide assignments passed this scoring filter, the corresponding MS͞MS spectra were manually reviewed. For an assign- ment to be accepted the MS͞MS spectrum must have a continuous b-ory-ion series of at least five residues and the three most intense fragment peaks assigned to ei- ther an a-, b-, or y-ion, to an a-, b-, or y-ion resulting from a neutral loss of water or ammonia, or to a multiply protonated fragment ion. All review of peptide assign- ments and manual interpretation of MS͞MS spectra were facilitated by SCAFFOLD, a proteome software package. PAPANO, a nitric oxide donor with defined release kinetics, or nonspecific labeling, ascorbate was omitted to largely prevent CysNO, an effective transnitrosating agent. Exposure of HASMC reduction of S-nitrosocysteine. Although ascorbate-independent to 100 ␮M CysNO for 20 min generated 3.0 Ϯ 0.3 nmol of protein biotin-HPDP labeling of S-nitrosocysteine is possible, naı¨ve and S-nitrosocysteine per mg of protein, whereas exposure to 2 mM cysteine-treated HASMC did not contain significant levels of PAPANO for 1 h generated 0.40 Ϯ 0.03 nmol of protein S- endogenous S-nitrosoproteins quantified by reductive chemistries nitrosocysteine per mg of protein (mean
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